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Animal Reproduction Science 100 (2007) 107117

Temporal effects of-tocopherol and l-ascorbicacid on in vitro fertilized porcine

embryo development

Mohammad Shamim Hossein, Md Abul Hashem, Yeon Woo Jeong,

Myeong Seop Lee, Sue Kim, Ji Hye Kim, Ok Jae Koo,Seon Mi Park, Eu Gine Lee, Sun Woo Park, Sung Keun Kang,

Byeong Chun Lee , Woo Suk Hwang

Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University,

56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea

Received 12 May 2006; received in revised form 17 June 2006; accepted 21 June 2006

Available online 24 July 2006

Abstract

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo

development. The present study evaluated temporaleffects of-tocopherol andl-ascorbic acid on the porcine

embryo development, and investigated whether a single or twice supplements of these two antioxidants at

a divided concentrations favors the embryo development. In order to determine temporal effects of -

tocopherol and/or l-ascorbic acid, 100M -tocopherol or 200M l-ascorbic acid were supplemented to

the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In

another set of experiments, the concentration was divided into two equal halves, i.e., 50 M -tocopherol

and 100M l-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture.

Supplementing culture media with 100M -tocopherol for the entire culture period of 168 h or starting

from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting fromthe 96 or 120 h of culture. l-Ascorbic acid (200M) alone or together with -tocopherol (100M) with

a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst.

l-Ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control.

No synergistic effect was observed on embryo development at a single supplement of these antioxidants.

Although, at divided supplements higher blastocyst percentage was observed compared with control group,

no further beneficial effect was observed compared with -tocopherol or l-ascorbic acid alone. Our results

demonstrated that the embryotrophic effects of-tocopherol and/or l-ascorbic acid, in terms of frequency

Corresponding author. Tel.: +82 2 880 1247; fax: +82 2 884 1902.

E-mail address: [email protected] (B.C. Lee).

0378-4320/$ see front matter 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.anireprosci.2006.06.013
mailto:[email protected]://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013mailto:[email protected]

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108 M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117

of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation

timing.

2006 Elsevier B.V. All rights reserved.

Keywords: -Tocopherol; l-Ascorbic acid; Porcine; IVF; Embryo culture

1. Introduction

Aerobic organism produces a range of reactive oxygen species (ROS) during metabolic pro-

cess (Nasr-Esfahni and Johnson, 1991; Gardner and Lane, 2002). The oocytes and embryos

also produce endogenous ROS by various enzymatic actions and metabolic pathways (Riley and

Behrman, 1991; Harvey et al., 2002). ROS participates in various cell processes especially in

tissue remodeling, hormone signaling, steriodogenesis and germ cell function at a physiolog-

ical concentration. However, it modifies normal cell functions, endanger cell survival, or bothwhen the critical concentration is overwhelmed (Droge, 2002). Therefore, ROS must be con-

tinuously inactivated in order to keep only a small amount necessary to maintain normal cell

functions.

Mechanisms for detoxification of and protection against ROS exit in in vivo system. These

include enzymes such as superoxide dismutase which produces hydrogen-peroxide from super-

oxide radicals, and catalase and glutathione peroxidase which decompose hydrogen-peroxide to

water (Guerin et al., 2001; Oyawoye et al., 2003). In addition, there are low molecular weight

antioxidants which act as ROS scavengers such as l-ascorbic acid, uric acid, glutathione and

tocopherols (McEvoy et al., 2000; Sturmey and Leese, 2003). Both the follicular and oviductal

fluid is rich in oxygen scavengers which protect oocytes and embryos from oxidative damage

(Gardiner and Reed, 1995; Lapointe et al., 1998). When oocytes and embryos are cultured in

vitro, they are deprived from these natural defense systems. Therefore, it is crucial to protect them

against oxidative stress during in vitro culture by adding antioxidant into the culture media to

optimize embryo production.-Tocopherol is the predominant lipid-soluble antioxidant in animal cells. It protects cells from

oxygen radicals in vivo and in vitro (Chow, 1991). Ascorbic acid is a water-soluble vitamin which

has been considered to be the most important antioxidant in extracellular fluids (Buettner, 1993;

Rose and Bode, 1993). Adding ascorbic acid into the culture media prevents follicular apoptosis

in rat and mouse follicles, and also improves the blastocyst production in mouse ( Eppig et al.,2000; Tilly and Tilly, 1995). Ascorbic acid acts synergistically with -tocopherol under some

conditions by regenerating tocopherol from tocopheroxyl radicals, the products of tocopherol and

free radical interaction (Chow, 1991).

Rapid changes on the cell physiology and embryonic metabolism intervene during the early

embryonic development (Gardner and Lane, 2002). The zygote has a low metabolic activity

and exhibits low levels of oxygen consumption whereas after blastocoel formation embryo

metabolism significantly increases (Harvey et al., 2002; Leese, 1995). So, the level of ROS

production may vary in different developmental stages as ROS produces along with metabolic

process. The susceptibility of embryos to ROS also varies according to developmental stages

(Ali et al., 2003). Thus, the requirement of antioxidant supplement into culture medium maynot be the same at different stages of development. There are reports about embryotrophic effect

of l-ascorbic acid and -tocopherol (Olson and Seidel, 2000; Wang et al., 2002; Kitagawa et

al., 2004) but no information is available about temporal effects of antioxidant supplement into
http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013http://dx.doi.org/10.1016/j.anireprosci.2006.06.013

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M.S. Hossein et al. / Animal Reproduction Science 100 (2007) 107117 109

the culture medium. Accordingly, the present study was undertaken to evaluate temporal effects

of-tocopherol and l-ascorbic acid on in vitro fertilized porcine embryo development, and to

investigate whether a single or two time supplement at divided concentrations favors the embryo

development.

2. Materials and methods

2.1. Recovery and culture of oocytes

Ovaries were collected from gilts at a local slaughterhouse and transported to the laboratory in

physiological saline supplemented with 100 U/ml penicillin G (SigmaAldrich Corp., St. Louis,

MO) and 100 mg/ml streptomycin sulphate (SigmaAldrich) at around 3035 C within 2 h of

collection. Cumulusoocyte complexes (COCs) from follicles 36 mm in diameter were aspirated

using an 18-gauge needle attached to a 10 ml disposable syringe (Kimetal.,2004). Compact COCswere selected and cultured in tissue culture medium (TCM)-199 (Invitrogen, Carlsbad, CA),

supplemented with 10 ng/ml epidermal growth factor (EGF; SigmaAldrich), 4 IU/ml pregnant

mare serum gonadotropin (PMSG) and hCG (Intervet, Boxmeer, Netherland) and 10% porcine

follicular fluid (pFF). The pFF was aspirated from 3 to 7 mm follicles from the prepubertal

gilt ovaries. After centrifugation at 150 g for 30 min, supernatant was collected and filtered

sequentially through 1.2 and 0.45m syringe filters (Gelman Sciences, Ann Arbor, MI). Prepared

pFF was stored at 20 C until use. Each group of 50 COCs was cultured in 500 l TCM-199

incubated at 39 C in a humidified atmosphere of 5% CO2 in 95% air. After culturing for 22 h,

COCs were washed three times and cultured in PMSG- and hCG-free TCM-199 medium for

another 22 h.

2.2. In vitro fertilization and culture

Frozen semen was thawed at 39 C for 1 min in a water bath, diluted in 10 ml Dulbeccos

PBS (Invitrogen) supplemented with 0.1% BSA (SigmaAldrich), 75g/ml potassium penicillin

G and 50g/ml streptomycin sulphate and centrifuged twice at 350 g for 3 min. Then, the

sperm pellet was suspended in modified Tris-buffered medium (mTBM) to give a concentra-

tion of 2 107 sperm/ml. The mTBM consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2,

20 mM Tris (T-1410; SigmaAldrich), 11 mM glucose, 5 mM sodium pyruvate and 8 mg/ml BSA(A-6003; SigmaAldrich). At 44 h of maturation culture, oocytes were freed from cumulus cells

by repeated pipetting in 0.1% hyaluronidase (SigmaAldrich) in TCM-199 medium and washed

three times with pre-equilibrated mTBM. After washing, 2025 oocytes were placed in 45 l

drops of the mTBM (fertilizing drop) covered with pre-warmed paraffin oil and 5 l sperm sus-

pension added to each fertilization drop to give a final sperm concentration of 2 106 sperm/ml.

After co-incubation of gametes for 6 h, the presumptive zygotes were washed and transferred

into in vitro culture (IVC) medium. The basic IVC medium was North Carolina State Univer-

sity (NCSU)-23 medium supplemented with 0.4% BSA (SigmaAldrich) (Kim et al., 2004).

Oocytes were cultured in NCSU-23 medium containing 0.5 mM pyruvate and 5.0 mM lac-

tate instead of 5.5 mM glucose at 39

C, 5% CO2, 5% O2 and 90% N2 atmosphere. At 96 hafter culture, the IVC medium was supplemented with 10% FBS (Invitrogen). The cleavage

rate and formation of blastocysts were evaluated under a stereomicroscope at 48 and 168 h of

culture.

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2.3. Differential staining

The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM)

and trophectoderm (TE) cells according to the modified staining procedure of Thouas et al.

(2001). Briefly, hatched blastocysts were used as such and non-hatched blastocysts were treatedwith 0.25% pronase (w/v, SigmaAldrich) for 5 min to dissolve the zonae pellucidae. After

rinsing in NCSU-23 medium, zona-free blastocysts were stained with 0.01% (w/v) bisbenz-

imide for 1 h. After rinsing in NCSU-23 medium, blastocysts were treated with 0.04% (v/v)

Triton X-100 (SigmaAldrich) for 3 min followed by treatment with 0.005% (w/v) propidium

iodide (SigmaAldrich) for 10 min. After rinsing with NCSU-23 medium, stained blastocysts

were mounted on glass slides under a cover slip and examined under an inverted micro-

scope (Nikon Corp., Tokyo, Japan) equipped with epifluorescence. The ICM nuclei labeled

with bisbenzimide appeared blue and TE cell nuclei labeled with both bisbenzimide and pro-

pidium iodide appeared pink. Any blastocysts without dual stain were excluded from the

study.

2.4. Experimental design

-Tocopherol and l-ascorbic acid were purchased from SigmaAldrich Corp. The drugs were

added into the NCSU-23 culture medium according to experimental designs. -Tocopherol was

first dissolved in 95% ethanol as a 2000 stock solution, stored in at 4 C wrapping with

aluminum foil and then diluted as a 10 concentration prior to each experiment. Ethanol

at a concentration of 0.05%, the concentration used to dissolve the -tocopherol, has no

effect on embryo development (Olson and Seidel, 2000). In order to determine the temporal

effects of -tocopherol and/or l-ascorbic acid, 100M -tocopherol and 200M l-ascorbic

acid was added to the culture media at 0, 48, 96 and 120 h of culture. To evaluate the

effects of two time supplement of -tocopherol and/or l-ascorbic acid at a lower concentra-

tion, the total concentration was divided into two equal halves, i.e., 50 M -tocopherol and

100M l-ascorbic acid and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of

culture.

2.5. Statistical analysis

In each experimental group, oocytes were randomly distributed. Each experiment was repeatedsix times. All data were subjected to one-way ANOVA followed by Tukey test to determine

differences among experimental groups using GraphPad Version 4.0 (Motulsky, 2003). Statistical

significance was determined when a P value was less than 0.05.

3. Results

3.1. Temporal effects of-tocopherol on in vitro fertilized porcine embryo development and

quality

Supplementing NCSU-23 with 100M -tocopherol for the entire culture period of 168 h(16.2%) or at 48 h of culture (17.3%) significantly increased the number of blastocyst percentage

compared with the control (10.5%) or supplementing at 96 h (9.6%) or 120 h (9.9%) of culture

(Table 1). Increased number (P < 0.05) of ICM, TE and total cells was observed when-tocopherol

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Table 1

Temporal effects of 100M -tocopherol on in vitro fertilized porcine embryo development and cell allocation to inner

cell mass (ICM) and trophectroderm (TE) in blastocysts

Duration

(h)

Embryo development Cell numbers (meanS.E.M.)

No. ofoocytes

No. oftwo-cell

embryos (%)a

No. ofblastocysts

(%)a

No. ofevaluated

blastocysts

ICM TE Total

Control 277 174 (62.8) 29 (10.5) a 18 11.2 0.3 a 30.1 0.8 a 41.2 1.1 a

0168 265 170 (64.2) 43 (16.2) b 26 12.9 0.4 bc 38.8 1.4 b 51.6 1.8 b

48168 272 185 (68.0) 47 (17.3) b 29 13.7 0.5 c 39.3 1.6 b 53.1 2.0 b

96168 260 175 (67.3) 25 (9.6) a 19 11.3 0.3 ab 31.4 1.0 a 42.7 1.1 a

120168 262 168 (64.1) 26 (9.9) a 17 11.4 0.4 ab 31.3 1.1 a 43.8 1.4 a

Within the same column, values with different letters (ac) were significantly different (P < 0.05).a Percentage of the number of oocytes.

was supplemented for the entire culture period of 168 h (12.9, 38.8 and 51.6, respectively) or at

48 h of culture (13.7, 39.3 and 53.1, respectively) compared with other experimental groups

(11.211.4, 30.131.4 and 41.243.8, respectively).

3.2. Effects of two time supplements of-tocopherol at a divided concentration on in vitro

fertilized porcine embryo development and quality

Higher blastocyst percentage (P < 0.05) was observed in all two time -tocopherol sup-plemented groups at a divided concentration (16.618.1% versus 9.4%) compared to the

control (Table 2). The increased number of total cells (54.057.2 versus 41.0), ICM

(13.814.5 versus 10.9) and TE (40.242.7 versus 30.1) cells was observed in the treat-

ment groups compared with the control. There was no difference in the frequency of blasto-

cyst formation and number of cells in blastocyst among different -tocopherol supplemented

groups.

Table 2

Effects of twotime supplement of-tocopherol at a divided concentrationon in vitrofertilized porcine embryo developmentand cell allocation to inner cell mass (ICM) and trophectroderm (TE) in blastocysts

Supplement

(h)


No. of

oocytes

No. of

two-cell

embryos (%)a

No. of

blastocysts

(%)a

No. of

evaluated

blastocysts

ICM TE Total

Control 220 170 (77.3) 22 (9.4) a 16 10.9 0.6 a 30.1 1.4 a 41.0 2.0 a

0 and 48 268 196 (73.1) 48 (18.1) b 26 13.8 0.6 b 40.2 2.4 b 54.0 3.0 b

0 and 96 280 212 (75.7) 48 (17.1) b 30 14.5 0.5 b 42.7 1.9 b 57.2 2.4 b

48 and 96 289 215 (74.4) 49 (16.6) b 32 13.9 0.5 b 40.9 2.2 b 54.9 2.7 b

Within the same column, values with different letters (a and b) were significantly different (P < 0.05). Total concentration

of-tocopherol (100M) were divided into two equal halves (50 and 50 M) and supplemented to the culture media at

different time.a Percentage of the number of oocytes.

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Table 3

Temporal effects of 200M ofl-ascorbic acid on in vitro fertilized porcine embryo development and cell allocation to

inner cell mass (ICM) and trophectroderm (TE) in blastocysts

Duration

(h)


No. ofoocytes

No. oftwo-cell

embryos (%)a

No. ofblastocysts

(%)a

No. ofevaluated

blastocysts

ICM TE Total

Control 252 170 (67.5) 24 (9.5) 14 11.3 0.4 30.7 1.3 42.0 1.6

0168 288 187 (64.9) 26 (9.0) 17 11.8 0.3 33.7 0.7 45.4 0.8

48168 270 185 (68.5) 28 (10.4) 24 11.6 0.3 33.1 0.5 44.8 0.7

96168 230 149 (64.8) 22 (9.6) 18 11.5 0.3 32.4 0.8 43.9 0.9

120168 240 167 (69.6) 24 (9.4) 19 11.9 0.4 33.5 1.8 45.4 2.1

a Percentage of the number of oocytes.

3.3. Temporal effects ofl-ascorbic acid on in vitro fertilized porcine embryo development

and quality

Supplementing 200M l-ascorbic acid into culture medium had no effect on the frequency

of blastocyst formation compared with the control (Table 3). No significant difference was

observed in the number of total cells (43.945.4 versus 42.0), ICM (11.511.9 versus 11.3)

and TE (32.433.7 versus 30.7) cells in treatment groups compared with the control.

3.4. Effects of two time supplement ofl-ascorbic acid at a divided concentration on in vitro

fertilized porcine embryo development and quality

Two time supplement of l-ascorbic acid at a divided concentration increased (P < 0.05) the

frequency of blastocyst formation (14.018.2% versus 8.9%) compared with the control (Table 4).

The increased number (P < 0.05) of total cells (52.458.2 versus 41.4), ICM (13.915.6 versus

11.1) and TE (38.542.6 versus 30.3) cells was observed in treatment groups compared with the

control. There were no differences in the frequency of blastocyst formation and number of cells

among l-ascorbic supplemented groups.

Table 4

Effects of two time supplement ofl

-ascorbic acid at a divided concentration on in vitro fertilized porcine embryodevelopment and cell allocation to inner cell mass (ICM) and trophectroderm (TE) in blastocysts

Supplement

(h)


No. of

oocytes

No. of

two-cell

embryos (%)a

No. of

blastocysts

(%)a

No. of

evaluated

blastocysts

ICM TE Total

Control 226 160 (70.8) 20 (8.9) a 16 11.1 0.5 a 30.3 1.0 a 41.4 1.5 a

0 and 48 268 196 (73.1) 46 (17.2) b 28 13.9 0.4 b 38.5 1.8 b 52.4 2.1 b

0 and 96 280 210 (75.0) 51 (18.2) b 32 15.6 0.5 b 42.6 1.7 b 58.2 2.1 b

48 and 96 279 208 (74.6) 44 (14.0) b 32 14.0 0.5 b 39.9 2.1 b 53.9 2.5 b

Within the same column, values with different letters (a and b) were significantly different (P < 0.05). Total concentration

ofl-ascorbic acid (200M) were divided into two equal halves (100 and 100M) and supplemented to the culture drop

at different time.a Percentage of the number of oocytes.

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Table 5

Temporal effects of simultaneous supplement of 100M -tocopherol and 200M l-ascorbic acid on in vitro fertilized

porcine embryo development and cell allocation to inner cell mass (ICM) and trophectroderm (TE) in blastocysts

Duration

(h)


No. ofoocytes

No. oftwo-cell

embryos (%)a

No. ofblastocysts

(%)a

No. ofevaluated

blastocysts

ICM TE Total

Control 186 130 (69.1) ab 19 (10.2) 14 12.9 0.5 32.4 0.7 45.1 1.0

0168 230 145 (63.0) b 22 (9.6) 11 11.6 0.5 30.9 1.4 42.5 1.5

48168 254 182 (71.7) a 38 (14.9) 22 12.1 0.3 32.1 0.1 44.2 0.9

96168 225 151 (67.1) ab 25 (11.1) 18 12.1 0.4 28.9 1.3 41.0 1.4

120168 230 150 (65.7) ab 27 (10.4) 16 11.9 0.3 29.8 1.7 41.7 1.9

Within the same column, values with different letters (a and b) were significantly different (P < 0.05).a Percentage of the number of oocytes.

3.5. Temporal effects of simultaneous supplement of-tocopherol andl-ascorbic acid on in

vitro fertilized porcine embryo development and quality

Simultaneous supplement of 100M -tocopherol and 200M l-ascorbic acid at a single

time did not improve the frequency of blastocyst formation (9.614.9% versus 10.2%) compared

with the control (Table 5). No significant difference was observed in the number of total cells

(41.044.2 versus 45.1), ICM (11.612.1 versus 12.9) and TE (28.932.1 versus 32.4) cells in

treatment groups compared with the control.

3.6. Effects of simultaneous two time supplements of-tocopherol andl-ascorbic acid at a

divided concentration on in vitro fertilized porcine embryo development and quality

Two time supplements of-tocopherol andl-ascorbic acid at a divided concentration improved

(P < 0.05) the number of blastocyst percentage (17.221.7% versus 10.3%) compared with the

control (Table 6). The increased number (P < 0.05) of total cells (55.462.4 versus 42.9), ICM

(16.117.3 versus 12.0) and TE (39.345.1 versus 30.9) cells was observed in treatment groups

compared with the control.

Table 6Effects of simultaneous two time supplement of-tocopherol and l-ascorbic acid at a divided concentration on in vitro

fertilized porcine embryo development and cell allocation to inner cell mass (ICM) and trophectroderm (TE) in blastocysts

Supplement

(h)


No. of

oocytes

No. of

two-cell

embryos (%)a

No. of

blastocysts

(%)a

No. of

evaluated

blastocysts

ICM TE Total

Control 223 160 (71.7) 23 (10.3) a 16 12.0 0.5 a 30.9 1.6 a 42.9 2.1 a

0 and 48 227 165 (72.7) 39 (17.2) b 28 16.5 0.6 b 41.7 2.1 b 58.2 2.4 b

0 and 96 230 169 (73.5) 50 (21.7) b 34 17.3 0.5 b 45.1 1.8 b 62.4 2.1 b

48 and 96 238 169 (71.0) 46 (19.3) b 32 16.1 0.4 b 39.3 1.3 b 55.4 1.6 b

Within the same column, values with different letters (a and b) were significantly different ( P < 0.05). -Tocopherol

(50M) and l-ascorbic acid (100M) were simultaneously supplemented to the culture drop at different time.a Percentage of the number of oocytes.

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4. Discussion

The present study was undertaken to evaluate the suitable timing and duration of antioxidant

supplement into the in vitro fertilized porcine embryo culture medium to improve embryo devel-

opment and its quality. Two well-known antioxidants, lipidophillic -tocopherol and hydrophilicl-ascorbic acid, were tested for their embryotrophic effects. Our results demonstrated that both

the -tocopherol and l-ascorbic acid have embryotrophic effects, alone or together, depending

on supplement timing, duration and concentration. In the previous studies, concentrations of-

tocopherol used in in vitro embryo production were varied from 50 to 800M and l-ascorbic

acid up to 400M (Wang et al., 2002; Kitagawa et al., 2004). Kitagawa et al. (2004) reported

that 100M -tocopherol had embryotrophic effect on porcine IVF derived embryos. Indeed,

concentrations more than 400M -tocopherol induced a dose-dependent decrease in blastocyst

development and blastocyst cell number (Wang et al., 2002). Thus, in the present study, we used

100M of-tocopherol to evaluate its temporal effects on porcine embryo development. In addi-

tion, 200M l-ascorbic acid was selected because higher concentration than 200M producedtoxicity in mouse embryos (Wang et al., 2002) and bovine oocytes (Dalvit et al., 2005).

The antioxidant activity of-tocopherol in preventing free radical-initiated peroxidative tissue

damages is accepted by most investigators and is believed to be the primary free radical scav-

enger in mammalian cell membranes (Rose and Bode, 1993; Droge, 2002). Large amount of-tocopherol is present in the ovary and follicular fluid which implies its diversified actions on

the reproductive system (Attaran et al., 2000). Adding -tocopherol to the bovine embryo culture

medium improved developmental competence of embryo to theblastocyst stage (Olson andSeidel,

2000). In the present study, -tocopherol at a concentration of 100M had beneficial effects on

porcine IVF derived embryo development. With a single supplement at 0 or 48 h of the culture

period and twice supplement with divided concentrations (at 0 and 48, 0 and 96, or 48 and 96 h),

supplementing -tocopherol produced significantly higher blastocyst percentage compared with

the control. Porcine embryos contain a much higher concentration of fatty acids compared with

other mammalian embryos (McEvoy et al., 2000; Sturmey and Leese, 2003), they might be more

susceptible to lipid peroxidation. As lipid peroxidation occurs as a chain reaction, the reaction

expands to surrounding phospholipids. Therefore, -tocopherol can stop this chain reaction and

eventually lead to more blastocyst formation. Ascorbic acid is a potent direct antioxidant which

provides protection against endogenous oxidative DNA damage (Fraga et al., 1991). It has been

supplemented in IVM and IVC medium with much debate on its embryotrophic effects. Tatemoto

et al. (2001) used 250Ml

-ascorbic acids during IVM of porcine oocytes and reported that itdid not improve the developmental competence of porcine embryos after IVF. Whereas, Wang et

al. (2002) reported that adding ascorbic acid into an embryo culture medium significantly affects

embryo development in a dose dependant manner. Our study demonstrated that supplementing

200M l-ascorbic acid into embryo culture media as a single supplement for any duration neither

improved the frequency of blastocyst formation nor the blastocyst quality. However, when the

concentration was divided into two equal halves and added at 0 and 48, 0 and 96, or 48 and 96 h,

higher blastocyst percentage was observed compared with the control. Our results suggest the

importance of concentration and duration ofl-ascorbic acid for embryotropic effects.

The functional interrelation between -tocopherol and micronutrients, notably the l-ascorbic

acid has long been recognized (Chow, 1991). However, in the present study, no synergistic effectof -tocopherol and l-ascorbic acid on embryo development was observed. Although, when

these two oxidants were added twice at a divided concentration, higher blastocyst percentage was

observed compared with the control, but no further development was observed compared with

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-tocopherol or l-ascorbic acid alone. It is suggested that antioxidant therapy may have negative

and undesirable effects if a safety threshold dosage of antioxidants is surpassed. Likewise our

study, Olson and Seidel (2000) reported that co-supplemention of-tocopherol and l-ascorbic

acid at a concentration of 100M each has deleterious effect on bovine. In a recent study, Dalvit

et al. (2005) stated that when the maturation medium for bovine oocytes was supplemented with-tocopherol and ascorbic acid together, the number of blastocyst percentage was diminished

significantly compared with not only the control but also with -tocopherol or ascorbic acid

alone. Thus, the reason why we did not obtain the synergistic effect of-tocopherol and ascorbic

acid at a single supplement could be high concentrations of the two antioxidants that surpassed

the safety threshold dosage of antioxidants.

The ROS production by early embryos and their susceptibility to ROS varies with the stages

of development. ATP production in the early embryo relies mostly on glycolysis, with less than

30% being produced via oxidative phosphorylation (Trimarchi et al., 2000; Harvey et al., 2002).

This suggests that more anaerobic metabolism of the early embryos could help to minimize the

production of ROS by the mitochondria, and thus reduce the risk of oxidative stress. In this regard,no embryotrophic effect of antioxidants was observed in the present study; neither l-ascorbic

acid nor -tocopherol had improved the cleavage rate at single or a divided supplement. Cleavage

rate was even lower when they were supplemented together at 0 h of culture. This indicates that

before the two cells stage embryo does not produce much ROS or they are more stable to the

deleterious effect of ROS or both. Embryo metabolism increases sharply as the cell number

increases in it. Because ROS production is related with metabolism, it is likely to produce

more ROS at advance stage of embryonic development. There is report that mouse blastomere

(Nonogaki et al., 1994) and bovine 916 cells stage embryos are vulnerable to ROS compared

to other stages (Ali et al., 2003). In the present study, no improvement in blastocyst frequency

was observed when -tocopherol was supplemented after 96 h of culture. This result suggest that

irreversible damage to the cell organelles might occur before this period that antioxidant cannot

recovered when added at/after 96 h of culture. Thus, antioxidant is recommended to supplement

into the culture media within first 48 h of culture.

5. Conclusion

Embryotrophic effects of-tocopherol were observed when media was supplemented a single

time with 100M or two times with 50M depending supplementing timing. On the other hand,

200Ml

-ascorbic acid alone or with 100 M -tocopherol had no effect on porcine embryonicdevelopment with a single time supplement. Simultaneous two time supplements of-tocopherol

and l-ascorbic acid with a divided concentration supported better porcine embryo development

and increase the embryo quality compared with control, no further beneficial effect was observed

compared with -tocopherol and l-ascorbic acid. So, we recommend to supplement porcine

embryo culture medium with 100M -tocopherol within first 48 h of culture at a single

supplement, or 50M -tocopherol or 100M l-ascorbic acid at twice supplements at 0 and

96 h of culture.

Acknowledgements

This study was supported by grants from the Korean Ministry of Science and Technology (Top

Scientist Fellowship). The authors acknowledge a graduate fellowship provided by the Ministry

of Education through BK21 program.

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